A proband with chylomicronemia, pancreatitis, and non-insulin-dependent diabetes (NIDDM) bears two different mutations in exon 3 of the lipoprotein lipase (LPL) gene: a missense mutation, 75Arg-->Ser, inherited through the paternal line and a truncation, 73Tyr-->Ter, through the maternal line. NIDDM appeared to be independently segregating. The R75S mutant was studied in extracts and media from transfected COS-1 cells. Detectable amounts of catalytically competent R75S LPL suggested destabilization of the active homodimer as with exon 5 mutants (Hata et al. 1992. J. Biol. Chem. 267:20132-20139). Hydrolysis of a short-chain fatty acid ester indicated that R75S does not directly affect activation of LPL by apoC-II. Subjects with NIDDM and wild-type LPL, and nondiabetic middle-aged carriers of the 73Tyr-->Ter truncation had moderate hypertriglyceridemia (260-521 mg/dl) and reduced high density lipoprotein cholesterol. A maternal aunt with NIDDM carried the truncation. Her phenotype (triglycerides of 5,300 mg/dl, eruptive xanthomatosis, and recurrent pancreatitis) was as severe as that in homozygotes or compound heterozygotes. We conclude: (a) diabetic carriers of dysfunctional LPL alleles are at risk for severe lipemia; and (b) the physiologic defects in NIDDM may be additive or synergistic with heterozygous LPL deficiency.
Most missense mutations of the lipoprotein lipase (LPL) gene identified among LPL-deficient subjects cluster in a segment of the sequence that encodes the catalytic triad as well as functional elements involved in the activation of the lipase at lipid-water interfaces. Consequently, loss of activity may result either from direct alterations of such functional elements or from less specific effects on protein folding and stability. This issue was addressed by examining biochemical properties of four such variants (A176T, G188E, G195E, and S244T) in a heterologous expression system (COS-1 cells). Variant G195E (GGA----GAA) was previously unreported. In all instances, inactive enzyme was recovered in medium, albeit at reduced levels. Cellular synthesis and extracellular degradation were similar to those for wild type, suggesting that reduced secretion resulted from increased intracellular degradation. When cell extracts were subjected to heparin-Superose affinity chromatography followed by elution on a linear salt gradient, all variants exhibited a single, inactive, low affinity immunoreactive peak. By contrast, wild-type enzyme presented an additional, high affinity, active species, which we interpret as homodimeric enzyme. Substitution of the active-site serine (S132A) led to loss of activity but maintenance of the high affinity species. When large amounts of the G188E variant were applied to the column, small but significant amounts of high affinity, active enzyme were recovered. Systematic substitutions at residue 188 showed that only glycine could accommodate structural constraints at this position. We conclude that the mutations examined did not impart lipase deficiency by affecting specific functional elements of the enzyme. Rather, they appear to affect protein folding and stability, and thereby formation and maintenance of subunit assembly.
Title: Lipoprotein lipase deficiency resulting from a nonsense mutation in exon 3 of the lipoprotein lipase gene Emi M, Hata A, Robertson M, Iverius PH, Hegele R, Lalouel JM Ref: American Journal of Human Genetics, 47:107, 1990 : PubMed
In DNA from a male patient of German and Polish ancestry who has lipoprotein lipase deficiency, sequencing of all nine exons and intron-exon boundaries corresponding to the coding region of the lipoprotein lipase gene detected a C----T transition leading to the substitution of a stop signal for the codon that normally determines a glutamine at position 106 of the mature enzyme. Hybridization with allele-specific oligonucleotides at this position established that the patient was homozygous for this mutation. This mutation must lead to the synthesis of a sharply truncated protein, accounting for the enzymatic deficiency noted in the patient.
Cloning and sequencing of lipoprotein lipase (LPL) cDNA prepared from the adipose tissue of a patient with classical LPL deficiency revealed a G to A transition at nucleotide 818 in all sequenced clones, leading to the substitution of glutamic acid for glycine at residue 188 of the mature protein. Hybridization of genomic DNA with allele-specific oligonucleotides confirmed that the patient was homozygous for this mutation and revealed that carrier status for this mutation among relatives of the patient was significantly associated with hypertriglyceridemia. Assay of the patient's plasma for immunoreactive enzyme and activity demonstrated the presence of a circulating inactive enzyme protein, the concentration of which was further increased by injection of heparin. The mutant sequence was produced by oligonucleotide-directed mutagenesis, and both normal and mutant sequences were cloned into the expression vector pSVL and transfected into COS-1 cells. The normal sequence led to the in vitro expression of an enzyme that bound to heparin-Sepharose and had a specific catalytic activity similar to that of normal postheparin plasma enzyme. By contrast, the mutant enzyme expressed in vitro was catalytically inactive and displayed a lower affinity for heparin than the normal enzyme. We conclude that this single amino acid substitution leads to the in vivo expression of an inactive enzyme accounting for the manifestations of LPL deficiency noted in the patient.
Title: Compound heterozygote for lipoprotein lipase deficiency: Ser----Thr244 and transition in 3' splice site of intron 2 (AG----AA) in the lipoprotein lipase gene Hata A, Emi M, Luc G, Basdevant A, Gambert P, Iverius PH, Lalouel JM Ref: American Journal of Human Genetics, 47:721, 1990 : PubMed
Cloning and sequencing of translated exons and intron-exon boundaries of the lipoprotein lipase gene in a patient of French descent who has the chylomicronemia syndrome revealed that he was a compound heterozygote for two nucleotide substitutions. One (TCC----ACC) leads to an amino acid substitution (Ser----Thr244), while the other alters the 3' splice site of intron 2 (AG----AA). The functional significance of the Thr244 amino acid substitution was established by in vitro expression in cultured mammalian cells.
Familial lipoprotein lipase (LPL) deficiency is a rare genetic disorder accompanied by well-characterized manifestations. The phenotypic expression of heterozygous LPL deficiency has not been so clearly defined. We studied the pedigree of a proband known to be homozygous for a mutation resulting in nonfunctional LPL. Hybridization of DNA from 126 members with allele-specific probes detected 29 carriers of the mutant allele. Adipose tissue LPL activity, measured previously, was reduced by 50% in carriers, but did not reliably distinguish them from noncarriers. Carriers were prone to the expression of a form of familial hypertriglyceridemia characterized by increased plasma triglyceride, VLDL cholesterol and apolipoprotein B, and decreased LDL and HDL cholesterol concentrations. These manifestations were age modulated, with conspicuous differences between carriers and noncarriers observed only after age 40. Several noncarriers exhibited similar lipid abnormalities, but without the inverse relationship between VLDL cholesterol and LDL cholesterol noted among carriers. In addition to age and carrier status, the potentially reversible conditions, obesity, hyperinsulinemia and lipid-raising drug use were contributory. Thus heterozygous lipoprotein lipase deficiency, together with age-related influences, may account for a form of familial hypertriglyceridemia.
A monoclonal antibody to lipoprotein lipase (LPL) has been used in an enzyme-linked immunosorbent assay (ELISA) for LPL protein mass. Measurement of LPL immunoreactive mass in pre- and postheparin plasma distinguished three classes of abnormalities in patients with classical deficiency of lipoprotein lipase activity. The class I defect consisted of the absence of LPL immunoreactive homodimer in pre- and postheparin plasma compatible with a potential 'null allele'. Patients with a class II defect had almost no LPL immunoreactive mass in preheparin plasma but showed an increase in their LPL mass of 68 +/- 23 ng ml-1 (mean +/- SD) after heparin. Patients with the class III defect had considerable amounts of LPL immunoreactive material in preheparin plasma (159 +/- 190 ng ml-1). Heparin administration, however, caused very little additional release of LPL into the plasma (16 +/- 51 ng ml-1). Thus although both class II and class III patients had an LPL protein with abnormal catalytic activity, class III patients also appeared to have a defect in heparin binding of LPL. To test this hypothesis, postheparin plasma of classes II and III patients was analysed by heparin-Sepharose chromatography. In contrast to class II patients, the LPL immunoreactive mass of class III patients did not show affinity for the heparin and eluted in the column void volume, suggesting the class III defect is also associated with a defect in heparin binding.
The enzyme lipoprotein lipase plays a central role in the processing of energy in the form of calorically dense triglyceride. Classical LPL deficiency usually presents in childhood with the multiple manifestations related to chylomicronemia. Many patients with genetic variations have been noted who differ in one of many ways from the classical patients. With the development of techniques to measure enzyme mass and to study gene expression, the molecular defects in each of these families should become evident.
Title: Lipoprotein lipase from bovine milk. Isolation procedure, chemical characterization, and molecular weight analysis Iverius PH, Ostlund-Lindqvist AM Ref: Journal of Biological Chemistry, 251:7791, 1976 : PubMed
Lipoprotein lipase of high purity has been isolated from bovine milk by affinity chromatography on heparin-Sepharose, adsorption to Cgamma-aluminum hydroxide gel, and intervent dilution chromatography on heparin-Sepharose. Chemical analysis shows that the enzyme is a glycoprotein containing 8.3% carbohydrate. The monomer molecular weight, determined under reducing conditions in 6.6 M guanidine HCl by sedimentation equilibrium ultracentrifugation and analytical gel chromatrgraphy, is 48,300 and 50,800, respectively. Analyses of the sedimentation coefficient (SO20,w=5.40 S) and the diffusion coefficient (DO20,w=48.8 mum2/s) in a buffer of physiological pH and ionic strength yield a molecular weight of 96,900. In solution, the native enzyme thus appears to be a dimer of presumably identical subunits.
